PROJECT SUMMARY Signaling through store-operated Ca2+ channels (SOCs) is critical for many physiological processes including immune cell activation and differentiation. Accordingly, the loss of SOC function leads directly to a lethal severe combined immunodeficiency syndrome in humans. SOCs are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER), which causes the ER Ca2+sensor STIM1 to accumulate at ER-plasma membrane (PM) junctions where it binds and activates Orai1, the pore-forming subunit of the Ca2+release- activatedCa2+(CRAC)channel,triggeringCa2+entryintothecell.Ourlong-termgoalistounderstandin molecular detail the underlying mechanisms that control Ca2+ influx through CRAC channels. We have developed a number of new approaches to tackle these issues, including single-molecule tracking of STIM1 and Orai1, gene editing with CRISPR/Cas9 to label and mutagenize endogenous proteins, tandem concatemers of Orai1 that allow subunit-selective mutagenesis of the CRAC channel, and single-molecule FRET to probe conformational dynamics in a highly defined in vitro system. Over the next five years, we will apply these approaches to understand CRAC channel regulation in three areas. First, we aim to understand the mechanisms of native STIM1 and Orai1 localization and interaction at ER-PM junctions. Nearly all we know about the SOC mechanism is based on heterologous high-level overexpression of STIM1 and Orai1, which is likely to override many important regulatory mechanisms involving low amounts of accessory proteins. We will exploit gene editing techniques to label and modify endogenous STIM1 and Orai1 and study the factors that control the initial trapping of STIM1 by the PM, the residence time of Orai1 in junctions, and the stoichiometry and interaction kinetics of STIM1 and Orai1 at native junctions. Second, we will extend mechanistic studies of Ca2+-dependent inactivation (CDI), the predominant mechanism for feedback inhibition of CRAC channels. By subunit-selective mutagenesis of hexameric Orai1 concatemers we will characterize the interactions of STIM1 with the II-III intracellular loop and selected pore residues of Orai1 that drive conformational changes underlying COl. The third and major focus will be to identify the dynamic conformational changes that underlie activation of STIM1 and Orai1. By measuring single-molecule FRET of labeled STIM1 and Orai1 in vitro, we will identify the structures that keep STIM1 inactive and how they rearrange after store depletion to activate STIM1. The single-molecule approach will be widely applied to other questions such as the stoichiometry, dynamics and conformation of STIM1 binding to Orai1 as well as the conformational changes leading to Orai1 pore opening and the effects of purified accessory proteins thought to modulate STIM-Orai interactions in living cells. These studies have the potential to resolve many of the most difficult and important issues related to SOC activation, and may suggest new strategies for modulating calcium signals to provide new treatments for autoimmune and immunodeficiency syndromes.